Modulation of neuronal atrophy in Huntington's disease

亨廷顿病神经元萎缩的调节

基本信息

批准号：
10248302
负责人：
Junghee Lee
金额：
$ 27.56万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10248302
关键词：
3-Dimensional Actins Affect Animal Model Ankyrin Repeat Atrophic Autopsy Binding Biological Assay Biosensor Brain CAG repeat CASP3 gene Cell Line Cell Proliferation Cell Size Cell physiology Cells Cleaved cell Co-Immunoprecipitations Code Confocal Microscopy Corpus striatum structure Cross-Sectional Studies Cytoskeleton Development Exons F-Actin Fluorescence Resonance Energy Transfer Functional disorder GRLF1 gene Gene Mutation Genes Genetic Transcription Glutamine Guanosine Triphosphate Phosphohydrolases Human Huntington Disease Huntington gene Image Analysis Impairment In Situ Nick-End Labeling In Vitro Lead Measures Mediating Modeling Molecular Motor Activity Mus Mutation Neurites Neurodegenerative Disorders Neurons Outcome PH Domain Pathogenesis Pathologic Pathway interactions Patients Phenotype Plasmids Process Proteins Site Stains Stress Fibers Structure Survival Rate Tail Suspension Testing Therapeutic Transgenic Animals Transgenic Mice Transgenic Organisms Transmission Electron Microscopy Triplet Multiple Birth Western Blotting base image reconstruction in vivo inhibitor/antagonist live cell imaging migration motor behavior motor symptom mouse model mutant novel overexpression protein function restoration rho GTP-Binding Proteins rho GTPase-activating protein small hairpin RNA spatiotemporal therapeutic target time use

项目摘要

Project Summary/Abstract Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disease caused by a CAG triplet expansion mutation coding for glutamine in exon 1 of the Huntingtin (HTT) gene. Mutant HTT (mHTT) protein disrupts a number of molecular and cellular processes. The Ras-related Rho GTPases are molecular switches that regulate a number of processes, including cell proliferation, differentiation, migration, transcription, and actin dynamics. Long-term RhoA inactivation mediated by p190RhoGAP and a Rap-dependent RhoGAP (ARAP3) is essential for neurite outgrowth. In our preliminary studies using in vitro HD striatal cell lines and HD transgenic mice, we discovered striking abnormalities of the ARAP3-RhoA pathway that we found in human HD postmortem brain. Rho-GTPase activity was increased 24-fold in the striatal lysates of HD patients while ARAP3 (ArfGAP with RhoGAP Domain, ankyrin repeat and PH Domain 3), a negative regulator of RhoA, was significantly down regulated. ARAP3 overexpression in striatal cells restored neuronal size and function that were affected by a constitutively active mutant RhoA. Delivery of AAV-shRNA ARAP3 significantly exacerbated neuronal atrophy in the striatum of YAC128 mice. Based on these findings we propose a novel hypothesis that altered ARAP3 function and RhoA activity cause potentially reversible F-actin stress fiber formation and cytoskeletal disruption which in turn lead to neuronal atrophy and dysfunction in HD. To investigate whether impaired ARAP3-RhoA pathway underlies the cellular and molecular basis of neuronal atrophy that is reversible, we propose three specific aims: Aim 1: To investigate alteration of ARAP3 and RhoA levels in postmortem brains, transgenic animal models, and cell line models of HD. We will determine the spatiotemporal change of ARAP3, RhoA, and cytoskeleton structures in the striatum by using Western blot, qPCR, and confocal microscopy combined with 3-D reconstruction image analysis. Aim 2: To determine the relationship between ARAP3 and the RhoA pathway, and identify molecular and cellular mechanisms of neuronal atrophy in HD. We will use time-resolved fluorescence resonance energy transfer (FRET)-based RhoA biosensor and live cell imaging to identify how loss or gain of ARAP3 function affects the RhoA activity in HD striatal cells. Aim 3: To examine the in vivo effects of ARAP3 and RhoA on neuronal atrophy and function, motor activity, and survival in HD mouse models. We will perform cross sectional studies to determine the loss of ARAP3 function and the gain of RhoA function on motor symptoms and survival rates in HD mice. We will analyze F-actin stress fiber formation and DARPP32 activity in the striatal neurons of mice. We will further measure neuropathological changes such as neuronal size/number and mHTT aggregation. Our studies will identify novel molecular and cellular mechanisms of neuronal atrophy in the pathogenesis of HD and provide a therapeutic approach for HD.

项目概要/摘要亨廷顿病 (HD) 是一种由 CAG 三联体引起的常染色体显性神经退行性疾病亨廷顿蛋白 (HTT) 基因外显子 1 中编码谷氨酰胺的扩展突变。 Ras 相关的 Rho GTP 酶是分子开关。调节许多过程，包括细胞增殖、分化、迁移、转录和 p190RhoGAP 和 Rap 依赖性 RhoGAP 介导的长期 RhoA 失活。在我们使用体外 HD 纹状体细胞系和 HD 的初步研究中，(ARAP3) 对于神经突生长至关重要。我们在转基因小鼠中发现了在人类中发现的 ARAP3-RhoA 通路的显着异常 HD 死后脑部的 Rho-GTPase 活性在 HD 患者的纹状体裂解物中增加了 24 倍。 ARAP3（带有 RhoGAP 结构域、锚蛋白重复序列和 PH 结构域 3 的 ArfGAP）是 RhoA 的负调节因子纹状体细胞中 ARAP3 的过度表达显着下调，从而恢复了神经的大小和功能。受到 AAV-shRNA ARAP3 的组成型活性突变体 RhoA 的影响显着加剧。基于这些发现，我们提出了一个新的假设。改变 ARAP3 功能和 RhoA 活性导致潜在可逆的 F-肌动蛋白应力纤维形成和细胞骨架破坏，进而导致 HD 中的神经元萎缩和功能障碍。研究受损的 ARAP3-RhoA 通路是否是神经元的细胞和分子基础由于萎缩是可逆的，我们提出了三个具体目标：目标 1：研究 ARAP3 和死后大脑、转基因动物模型和 HD 细胞系模型中的 RhoA 水平。确定纹状体中 ARAP3、RhoA 和细胞骨架结构的时空变化蛋白质印迹、qPCR 和共聚焦显微镜与 3D 重建图像分析相结合目标 2：确定 ARAP3 和 RhoA 通路之间的关系，并鉴定分子和细胞我们将使用时间分辨荧光共振能量转移来研究HD中的神经萎缩机制。基于 FRET 的 RhoA 生物传感器和活细胞成像，以确定 ARAP3 功能的丧失或增强如何影响 HD 纹状体细胞中的 RhoA 活性目标 3：检查 ARAP3 和 RhoA 对神经元的体内作用。我们将在 HD 小鼠模型中进行萎缩和功能、运动活动和存活。研究确定 ARAP3 功能丧失和 RhoA 功能增强对运动症状和我们将分析纹状体中 F-肌动蛋白应激纤维的形成和 DARPP32 活性。我们将进一步测量小鼠神经元的大小/数量和 mHTT 等神经病理学变化。我们的研究将确定神经萎缩的新分子和细胞机制。探讨HD的发病机制并为HD的治疗提供方法。